Choline is indispensable for a number of fundamental processes in the body. Besides being the precursor of the acetylcholine, an important neurotransmitter, choline is found in the cell membrane structure combining with fatty acids, phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty acid build up in the liver, along with increased cholesterol levels, high blood pressure and memory loss. Thus, rapid detection methods are required for the determination of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination of choline. During construction of the biosensor, glucose oxidase was used as a model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v) in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a crosslinker in a series of membranes. HEMA-grafted membranes were activated with epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme. The enzyme activity was measured with an oxygen electrode unit based on oxygen consumption upon substrate addition. Membranes were characterized in terms of grafting conditions and mechanical properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS (0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical test results revealed that HEMA grafting made Teflon membrane more flexible and the presence of EGDMA made the grafted membrane stiffer. During optimization stage, it was found that the immobilized enzyme amount was not sufficient to obtain enzyme activity. Thus, the membrane preparation stage was modified to obtain thinner membranes. The immobilized glucose oxidase and choline oxidase contents on thin HEMA grafted membranes were determined by Bradford and Lowry methods. The influence of EGDMA presence and the epichlorohydrin activation duration on enzyme activity studies revealed that the membrane should be prepared in the absence of EGDMA and 30 min activation duration is appropriate for epichlorohydrin coupling. The study on the influence of membrane activation procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching membrane on the electrode directly rather than placing in the sample unit, the response of the enzyme immobilized sensor improved with high specific activity. The optimum choline oxidase concentration was found to be 2 mg/mL considering the effect of immobilization concentration on enzyme activity. With the choline oxidase biosensor, the linear working range was determined as 0.052-0.348 mM, with a 40 &plusmn / 5 &micro / M minimum detection limit. The response of the sensor decreased linearly upon successive measurements.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme.

D-Arginine dehydrogenase (DADH) catalyzes the oxidation of D-arginine to imino arginine using FAD as the cofactor. The enzyme is part of a recently discovered two-enzyme complex from Pseudomonas aeruginosa involved in arginine utilization. Function of the enzyme within the organism is unknown. Work on this enzyme has been undertaken to understand the structure as well as its reaction mechanism so as to eventually assign a function to the enzyme within the physiological context. In the reductive half-reaction 2 e- and 1 H+ are transferred from the amino acid substrate to FAD cofactor. In the oxidative half-reaction the reducing equivalents from the FAD cofactor are passed to an electron acceptor that is yet to be discovered. The enzyme has been established to have no reactivity with O2. Choline oxidase (CHO) from Arthrobacter globiformis is a well characterized member of Glucose-Methanol-Choline Superfamily that reacts with molecular O2. It catalyzes the oxidation of choline to glycine betaine mediated by betaine aldehyde intermediate using FAD as the cofactor and O2 as the oxidant to regenerate oxidized FAD for further reaction. Glycine betaine, the product of the reaction is an important osmolyte that regulates nutrients for plants under stressful conditions. Therefore it is of commercial interest to genetically engineer crops that do not typically possess competent pathways for glycine betaine synthesis. In this dissertation molecular details concerning the reductive half-eaction of DADH and oxidative half-reaction of CHO have been studied using a combination of steady state kinetics, rapid kinetics, pH, multiple substrates, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects or computational approaches. In DADH, the oxidation of amino acid substrate by FAD has been shown to most likely proceed via hydride transfer mechanism in the reductive half-reaction with Glu87, Tyr53, Tyr249 and His48 emerging as key players in substrate binding, catalysis or for up keeping the integrity of the FAD cofactor. In CHO, the oxidative half-reaction proceeds without stabilization of any reaction intermediates with H atom from reduced FAD and H+ from solvent or solvent exchangeable site occurring in the same kinetic step.

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 oC. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, a limited number of compounds that accumulate to high levels in cytoplasm to prevent dehydration and plasmolysis in adverse hyperosmotic environments. With this respect, the study of choline oxidase has potential for the development of therapeutic agents that inhibit the biosynthesis of glycine betaine, thereby rendering pathogenic bacteria susceptible to either conventional treatments or the immune system. In this study, the highly GC rich codA gene encoding for choline oxidase was cloned, expressed. The resulting enzyme was purified to high levels, allowing for detailed biochemical, mechanistic and structural characterizations. A chemical mechanism for the reaction catalyzed by choline oxidase was established by using kinetic isotope effects and viscosity effects as probes, in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage. Furthermore, these experiments indicated that chemical steps of flavin reduction by choline and betaine aldehyde are rate limiting for the overall turnover of the enzyme. Further mechanistic characterization clearly suggested a hydride transfer mechanism that is fully quantum mechanical. The structure of choline oxidase was resolved at 1.86 Å resolution in collaboration with the group of Dr. Allen O. Orville, at the Georgia Institute of Technology, providing a structural framework that is consistent with the mechanistic studies. The results of these studies will be presented and discussed in the context of the Glucose-Methanol-Choline oxidoreductase enzyme superfamily, of which choline oxidase is a member. Previous structural and mechanistic studies of alcohol- and aldehyde-oxidizing enzymes with different cofactors, as well as the biotechnological and biomedical relevance of choline oxidase are presented in Chapter 1. Chapter 3-8 illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter 3), spectroscopic and steady state kinetics (Chapter 4), the determination of the chemical mechanism for alcohol oxidation and the investigation of the involvement of quantum mechanical tunneling (Chapter 5 and 6), the study of aldehyde oxidation (Chapter 7), and the structural determination of choline oxidase by x-ray crystallography (Chapter 8). Chapter 9 presents a general discussion of the data presented.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented.

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions.

Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in choline oxidase have been investigated. Crystallographic studies of wild-type choline oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8á-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of choline oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of choline oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type choline oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in choline oxidase.

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine via the formation of an aldehyde intermediate. The oxidation of choline includes two reductive half-reactions followed by oxidative half-reactions. In the first oxidation reaction, the alcohol substrate is activated to its alkoxide via proton abstraction and oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. The role of Ser101 was investigated in this dissertation. Replacement of Ser101 with threonine, alanine, cysteine, or valine demonstrated the importance of the hydroxyl group of Ser101 in proton abstraction and in hydride transfer. Moreover, the kinetic studies on the Ser101Ala variant have revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase. The UV-visbible absorbance of Ser101Cys suggests Cys101 can form an adduct with the C4a atom of the flavin. The mechanism of formation of the C4a-cysteinyl adduct has been elucidated. D-arginine dehydrogenase (DADH) catalyzes the oxidation of D-amino acids to the corresponding imino acids, which are non-enzymatically hydrolyzed to α-keto acids and ammonia. The enzyme is strick dehrogenase and deoesnot react with molecular oxygen. Steady state kinetic studies wirh D-arginine and D-histidine as a substrate and PMS as the electron acceptor has been investigated. The enzyme has broad substrate specificity for D-amino acids except aspartate, glutamate and glycine, with preference for arginine and lysine. Leucine is the slowest substrate in which steady state kinetic parameters can be obtained. The chemical mechanism of leucine dehydrogenation catalyzed by DADH was explored with a combination of pH, substrate and solvent kinetic isotope effects (KIE) and proton inventories by using rapid kinetics in a stopped-flow spectrophotometer. The data are discussed in the context of the crystallographic structures at high resolutions (<1.3 Å) of the enzyme in complex with iminoarginine or iminohistidine.

ABSTRACT BIOACTIVE SURFACE DESIGN BASED ON CONDUCTING POLYMERS AND APPLICATIONS TO BIOSENSORS Ekiz, Fulya M. Sc., Department of Biotechnology Supervisor: Prof. Dr. Levent Toppare Co-Supervisor: Prof. Dr. Suna Timur June 2012, 88 pages An underlying idea of joining the recognition features of biological macromolecules to the sensitivity of electrochemical devices has brought the concept of biosensors as remarkable analytical tools for monitoring desired analytes in different technological areas. Over other methods, biosensors have some advantages including high selectivity, sensitivity, simplicity and this leads to solutions for some problems met in the measurement of some analytes. In this context, conducting polymers are excellent alternatives with their biocompatibility and ease of applicability for an efficient immobilization of biomolecules in preparing biosensors. Using several materials and arranging the surface properties of the electrodes, more efficient and seminal designs can be achieved. In this thesis, it is aimed to create new direct biosensors systems for the detection of several analytes such as glucose and pesticides thought to be harmful to the environment. Recently synthesized conducting polymers (polyTBT) / (poly(2-dodecyl-4,7-di(thiophen-2-yl)-2H-benzo[ d][1,2,3]triazole) and (poly(TBT 6 -NH2 ) / poly(6-(4,7-di(thiophen-2-yl)-2H-benzo[d][1,2,3]triazol-2-yl)hexan-1-amine) were utilized as a matrices for biomolecule immobilization. After successful electrochemical deposition the polymers on the graphite electrode surfaces, immobilization of glucose oxidase (GOx) and choline oxidase (ChO) were carried out. Amperometric measurements were recorded by monitoring oxygen consumption in the presence of substrates at -0.7 V. The optimized biosensors showed a very good linearity with rapid response times and low detection limits (LOD) to glucose and choline. Also, kinetic parameters, operational and storage stabilities were determined. Finally, designed biosensor systems were applied for glucose and pesticide detection in different media.